The goal of the work described in this proposal is to construct a quantitative and clinically useful assay to measure activity of multiple tyrosine kinases from cellular extracts of chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML) patients. Most CML cases are associated with a chromosomal translocation resulting in the consitutively active BCR-ABL tyrosine kinase while a large number of AML patients overexpress or have activating mutations in the FLT3 tyrosine kinase. Imatinib mesylate, an inhibitor of BCR-ABL, demonstrates tremendous efficacy in inactivating BCR-ABL in vivo and achieving remission in CML patients. However, certain BCR-ABL mutations are imatinib-resistant. Therefore, an in vitro measurement to assess the level of BCR-ABL activity and its dose-dependent inhibition upon treatment with imatinib would provide valuable information for tailoring individual CML patient treatment programs. Similarly, FLT3 kinase inhibitors are in clinical trials and resistant mutants are likewise expected to exist. BCR-ABL and FLT3 substrates will be synthesized and labeled with an acrylic moiety. Then these substrates will be copolymerized with acrylamide and crosslinker in an emulsion polymerization to generate microspheres via two-phase microfluidic flow. The microspheres will be exposed to extracts from cell lines expressing BCR-ABL and FLT3 and to white blood cell extracts derived from CML patients. Quantitative levels of substrate phosphorylation will be detected by anti-phosphotyrosine antibody labeling and flow cytometry. In addition, a method using thiophosphorylation and subsequent labeling of thiophosphates will be developed as an alternative to antibody labeling. Sensitivity and specificity of detection via antiphosphotyrosine antibodies and thiophosphorylation will be compared. The assay developed in this study will be compared to standard in vitro kinase assays as well as to methods currently used to diagnose CML, with respect to sensitivity and specificity. In this study our specific aims are: 1. Develop acrylamide-peptide copolymer microspheres for measuring Bcr-Abl activity in cell extracts 2. Quantify Bcr-Abl inhibition by imatinib mesylate in cell extracts and tissue samples from CML patients 3. Assess activity of multiple kinases using an acrylamide-peptide copolymer microsphere assay Relevance: The work described in this proposal will develop an assay to directly diagnose elevated protein tyrosine kinase activity related to chronic myeloid leukemia and predict how an individual patient will respond to drug therapy. This assay will likely be more rapid and less expensive than current methods, and have the advantage of predicting response to drug treatment. [unreadable] [unreadable] [unreadable]